Project Summary/Abstract The goal of this proposal is to understand nature's well-controlled chemistry that occurs in enzymes, by following the structural dynamics of the protein and chemical dynamics of the catalyst simultaneously. It is our goal to understand the design concepts from nature with X-ray crystallography and X-ray spectroscopy techniques using X-ray Free Electron Lasers (XFELs). Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving the reaction mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses from X-ray free electron lasers provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at synchrotron X-ray sources. The fs X-ray pulses make it possible to acquire the signal before the sample is destroyed. The objective of this proposal is to study the protein structure and dynamics of metallo-enzymes using crystallography, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism. We will design and apply a full suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods, that make use of the unique properties of the XFEL beam, to follow the reaction at room temperature. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both emission and absorption spectroscopy to get a complete understanding of the time-evolution of the electronic structure, while simultaneous room temperature time-resolved X-ray crystallography would provide the changes in the geometric structure of the overall protein. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology that use high-valent Fe and other elements for oxygen and C-H bond activation. We will focus on non-heme enzymes such as ribonucleotide reductase (Mn/Fe and Fe/Fe) and methane mono oxygenase (Fe/Fe), and heme containing Cyt P450 systems, and functional analogs of heme-copper oxidase systems engineered into simpler proteins.